The present invention relates to a novel carrier which may be sensitized with antigen and antibody and is capable of immobilizing enzyme. A method of preparing the carrier is also disclosed.
In the field of serological tests, indirect passive hemagglutination using an antigen-antibody reaction is widely employed to diagnose various diseases. This reaction is based on the fact that the antigen or antibody which is fixed on the surface of carrier particles reacts with the corresponding antigen or antibody in a sample serum. As a result, agglutination of the particle takes place.
Non-biological particles, such as polystyrene latex, kaolin and carbon powder and biological particles, such as mammalian erythrocytes and microbial cells may be used as carriers in the serological tests described above. Generally, non-biological particles are chemically stable, and are not antigenic. However, antigen and antibody are not tightly adsorbed on the non-biological carrier. For example, when the carrier having antigen or antibody absorbed thereon (sensitized carrier), is lyophilized in order to preserve it, the antigen or the antibody is released from the carrier particles. Consequently, the sensitized carrier should be preserved in the form of a suspension under low temperatures and in the absence of light. As a result, such sensitized carriers have a relatively short shelf life.
Kaolin and carbon powder suffer from the disadvantage in that it is difficult to obtain particles of equal size. Polystyrene latex is deficient because it tends to aggregate naturally in the neutral pH range where indirect passive agglutination is usually carried out.
On the other hand, mammalian erythrocytes and microbial cells are biological particles which have substantially uniform sizes. However, their size depends on the species of animal or microorganism, and accordingly, it is not always possible to obtain particles having the desired size. Among the biological carriers, mammalian erythrocytes are the most readily available and their sizes are uniform. However, their surfaces have specific antigenicity which can lead to nonspecific agglutination. Furthermore, it is difficult to obtain high quality erythrocytes due to variations of the biological, chemical and physical properties of the mammals from which the erythrocytes are obtained.
It has now been found that when the particles of gelatin, produced from a gelatin solution within a particular pH range containing a water-soluble polysaccharide and a sodium metaphosphate, are insolubilized with a cross-linking agent such as an aldehyde, the particles are suitable as a carrier for antigens and antibodies for indirect passive agglutination.
Any commercial gelatin material may be used in the invention. Among the most preferred commercial gelatin products are gelatins having an isoelectric point at a pH between about 8 and 9.
The water-soluble polysaccharide is used as a viscosity increasing agent or a paste, and includes derivatives and salts of such polysaccharides. Examples of such polysaccharides are arabic gum, carboxymethyl cellulose, sodium alginate, agar, and so on.
The sodium metaphosphate used in the invention is a compound having the formula of (NaPO.sub.3).sub.n, where n is a whole number from 3 to 6, and includes sodium trimetaphosphate and sodium hexametaphosphate.
The solution may also contain such other components as a water-miscible organic solvent, a nonionic or anionic surfactant, and a coloring agent.
The water-miscible organic solvent may optionally be added to accelerate the precipitation of the gelatin particles. Such solvents include lower alcohols, such as methanol, ethanol and isopropanol, and acetone. Since the organic solvent gives hydrophobicity to the particles, their physical and chemical properties differ somewhat from the particles which are made without the solvent.
The anionic surfactant and the nonionic surfactant may optionally be employed in order to prevent aggregation of the particles formed in the solution. The anionic surfactant includes alkylsulfosuccinic acids, alkylsulfomaleic acids, alkylsulfuric acid esters, and polyoxyethylene alkyl ether sulfuric acid esters. The nonionic surfactant includes polyoxyethylene fatty acid esters, polyoxyethylene alkyl ethers, polyoxyethylene alkyl phenyl ethers, and polyethylene glycol fatty acid esters wherein the alkyl group for both groups of surfactants has between 6 and 25 carbon atoms. Aggregation of the particles may also be prevented by chilling the solution below 10.degree. C. as soon as the particles form.
If it is desirable to color the carrier particles, a suitable coloring agent may be added to the solution. Alternatively, the insolubilized particles described below may be treated with the coloring agent. However, in the former case, the coloring agent is concentrated on the particles formed in the solution, thereby reducing the amount of the coloring agent needed. Moreover, the coloring agent is uniformly distributed to the inner part of the particles, and accordingly, the particles are tightly colored.
The coloring agents include red coloring matter, such as rhodamine, rose bengal, ponceau 3R, Bordeaux S, fuchsin, eosine, and neutral red, and blue coloring matter, such as crystal violet, toluidine blue, and methylene blue. However, reactive dyes, such as reactive red and reactive blue are most preferable, because the color of the carrier does not come off.
The concentration of the components in the solution are discussed below. All percentages are percentages by weight unless otherwise noted. The concentration of gelatin in the solution is about 0.01 to 2%, preferably about 0.05 to 1.0%, the concentration of the water-soluble polysaccharide is about 0.01 to 2%, preferably about 0.05 to 1.0%, and the concentration of sodium metaphosphate is about 3 to 15% by dry weight of the gelatin. The amount of the optional components may vary. The preferred concentration of the water-miscible organic solvent is about 4 to 25 vol%. The preferred concentration of the anionic surfactant is about 0.001 to 0.01%, and that of the nonionic surfactant is about 0.01 to 0.1%. The concentration of the coloring agent is usually about 0.005 to 0.5%.
The process of preparing the solution is not limited, and for example, each component may be separately dissolved in warm water and then the resulting solutions are mixed. Alternatively, all components may be placed in a vessel and then dissolved. However, it is preferable that the water-miscible organic solvent is added as one of the last components to make a suitable dispersion of the other components. Further, it is preferrable to separately dissolve the water-soluble polysaccharide because the water-soluble polysaccharide often contains a small amount of insoluble components.
If the pH of the solution is lower than the isoelectric point, white turbidity results when the gelatin is reacted with the water-soluble polysaccharide. Since this white turbidity is undesirable, the turbidity must be removed by the addition of alkali prior to the pH adjustment described below. Accordingly, when gelatin having an isoelectric point at a pH of about 8 to 9 is employed, alkali is preferably added to the solution until the pH of the solution reaches within a range around the isoelectric point.
The temperature of the solution should be higher than the temperature of gelation of the gelatin. The temperature of gelation depends on the concentration of the gelatin, etc., and it is usually between about 25.degree. and 50.degree. C.
Subsequently, the pH of the solution is adjusted to 2.5 to 6.0 by adding acid while the solution is stirred. During the pH adjustment, particles are produced in the solution. In order to produce uniform particles, the acid is added dropwise to the solution, while the solution is heated at between about 35.degree. and 50.degree. C. under moderate stirring. The optimal pH within the range of pH 2.5 to 6.0 depends on the desired size of particles and composition of the solution. For example, when the particles are employed as the carrier for indirect passive agglutination, the suitable size of the carrier is between about 2 and 10 microns, and in this case, the optimal pH is in the range of 4.0 to 5.5. The acid employed for the pH adjustment is not limited, and either an inorganic acid or organic acid may be employed. However, a weak acid, such as acetic acid, is preferred.
There is no equilibrium relation between the particles formed by the pH adjustment and the mother liquor, because the particles do not disappear when the temperature of the solution is lowered below the temperature of gelation. The particles are usually positively charged, through it depends on the ratio of gelatin to water-soluble polysaccharide. On the surface of the particles, metaphosphate ion is oriented and electric double layer is constructed around the particles. This electric double layer makes the suspension of the particles stable.
After the addition of the acid, the suspension of the particles is immediately cooled below 10.degree. C. in order to prevent aggregation of the particles. Then, an aldehyde cross-linking agent is added to the suspension, and the particles are insolubilized by standing overnight at a temperature below 10.degree. C. The amount of the cross-linking agent is between about 0.1 and 200% by dry weight of gelatin. The cross-linking agent is preferably glutaraldehyde, formaldehyde, glyoxal, crotonaldehyde, acrolein, and acetoaldehyde. Glutaraldehyde is the most preferred.
After treatment with the cross-linking agent, the particles are recovered by means of centrifugation, etc., and washed twice or three times with water containing surfactant, if necessary. In this case, the same surfactant as employed for dispersing the particles may be used at the same concentration.
The insolubilized particles thus produced may be used as a carrier for various purposes. However, the particles sometimes swell in a salt solution, and accordingly, it is preferable that the particles are further treated with the aldehyde cross-linking agent. For example, when the carrier is sensitized with antigen in a phosphate buffer solution, the carrier is preferably treated with formalin under the same conditions as the case of erythrocytes. By treatment with formalin, swelling of the carrier is alleviated, and the carrier may be preserved for a long term because of the sterilizing effect of formalin.
The carrier of the invention can immobilize antigen, antibody, enzyme and so on. As to the method of immobilization, for example, when antigen or antibody is sensitized on the carrier, the sensitization may be carried out according to conventional sensitization procedures using mammalian erythrocytes as the carrier. Such immobilization methods include the method of using tannic acid, the method of using formalin, the method of using glutaraldehyde, the method of using bisdiazotized benzidine, the method of using pyruvic aldehyde, the method of using toluene-2, 4-diisocyanate, etc.
The capability of the carrier of the invention is almost equal to that of mammalian erythrocytes which are considered the best carrier for indirect passive agglutinaton. Moreover, the present carriers are superior to the mammalian erythrocytes, because they are chemically and physically uniform and stable, and have no antigenic activity. And, according to the method of the invention, the carrier having the desired size is easily and inexpensively produced on a large scale.